Projects

1. Effect of cytidine deaminase on HIV RTC structure and function

Jill Carr, Adam Davis, Chris Burrell and Peng Li

The cytidine deaminase APOBEC3G has been identified as a cellular factor that can restrict HIV infection under certain circumstances. APOBEC3G has been shown to deaminate cytidine residues on the newly synthesised minus strand HIV DNA, thus inducing lethal G-A hyper-mutations in the HIV genome. Thus, APOBEC3G is an exciting innate defence mechanism that may be exploited in a therapeutic way to prevent HIV infection. However, recent studies have suggested that cytidine deaminase (CD) activity of APOBEC3G is not always strictly associated with APOBOEC3G anti-viral activity (see references). In our laboratory we have been studying components and effectors of HIV reverse transcription complexes (RTCs) – nucleoprotein complexes that are the site of active HIV reverse transcription in newly infected cells. We have observed that, when APOBEC3G is packaged into virions, the RTCs in newly infected cells are structurally altered and hypermutated by both APOBEC3G and potentially other mutators (Carr et al., manuscript in preparation). Whether this effect is a consequence of CD activity or secondary to structural alterations in the RTC is unknown. In this study we aim to investigate the ability of cytidine deaminase activity within the RTC to induce structural changes in RTCs and mutations in RTn products.

Vpr is an HIV accessory protein that is known to be part of the RTC and has previously been ‘tagged’ with other protein fusions without deleterious effects on HIV infection. This project will generate Vpr fusions with the APOBEC3G CD domain and analyse (i) CD activity of the fusion protein (ii) infectivity of virus carrying the fusion protein and (iii) physical properties of RTCs and mutation levels in RTn products made following infection with this virus.

The student will expect to gain experience in cloning, general molecular biology, cell culture, sucrose gradient sedimentation and techniques for analysis of viral replication such as ELISA to quantitate viral protein released, real time PCR to quantitate viral DNA. All infectious work will be performed by other trained personnel in the laboratory. Results from this project will provide novel data on the modes of actions of APOBEC3G that will increase our understanding of this new and exciting area.

Funding Sources

NHMRC, Australian Centre for HIV and Hepatitis Virology Research